Method for producing a nematocidal composition by heat treating a pH-adjusted fermentation broth

ABSTRACT

The present invention is directed to a method for producing a new nematocidal composition particularly useful against plant parasitic nematodes and also a process to prevent damage resulting from nematode infestation. The method for production of the composition involves heating a pH-adjusted fermentation broth of microorganisms to a temperature of at least about 100° C. for at least about 15 minutes. Preferably, the microorganism is  Gibberella fujikuroi, Streptomyces erythraeus, Bacillus sphaericus, Bacillus thuringiensis  or  Fusarium moniliforme.

FIELD OF THE INVENTION

[0001] The present invention is directed to a method for producing a newnematocidal composition particularly useful against plant parasiticnematodes and also a process to prevent damage resulting from nematodeinfestation. The method for production of the composition involvesheating a pH-adjusted fermentation broth of microorganisms to atemperature of at least about 100° C. for at least about 15 minutes.

BACKGROUND OF THE INVENTION

[0002] Plant parasitic nematodes cause serious economic damage to manyagricultural crops around the world. The nematodes in this group aremicroscopic worms and are, in general, obligate parasites of plants.They feed mostly on the roots of host plants;

[0003] however, several genera are known to parasitize above-groundparts including stems, leaves and flowers as well. Almost all the plantspecies of economic importance are susceptible to infection by somespecies of nematodes (notable exceptions are in the marigolds andasparagus). For example, root knot nematodes (RKN), (Meloidogyne spp.)are capable of parasitizing more than 3,000 species of crop plants.These plants include agronomic crops, vegetables, fruits, floweringtrees and shrubs. Nematodes reportedly cause crop loss equivalent tomore than six billion dollars in the United States alone and more thanone hundred billion dollars around the world.

[0004] The symptoms due to parasitic nematode injury vary widelydepending on the plant host, the nematode species, age of the plant,geographical location and climatic and external environmentalconditions. In general, an overall patchy appearance of plants in afield is considered indicative of nematode infestation. Morespecifically, nematode injury results in galling of the roots (abnormalswelling in the tissue due to rapid multiplication of cells in thecortical region) caused by species of root knot (Meloidogyne spp.) andcyst (Heterodera spp.) nematodes, lesions (localized, discolored areas)caused by lesion nematodes (Pratylenchus spp.), suppression of celldivision resulting in stubby roots (Trichodorus spp.), growthabnormalities including crinkling or twisting of above-ground parts(Aphelenchoides spp.), and even cell necrosis (death) in some cases.Plant parasitic nematodes may be endoparasitic in nature, as in the caseof the root-knot and lesion nematodes, or ectoparasitic as in the daggernematode (Xiphinema spp.) and lance nematode (Hoplolaimus spp.).Nematodes can be vectors of plant viruses and are also known to inducedisease complexes predisposing plants to infection by other plantpathogenic fungi and bacteria.

[0005] Chemical nematocides, either soil fumigants or non-fumigants,have been in use for many years and are among the few feasible optionsfor countering nematodes. At present, the process involves repeatedapplications of synthetic chemicals to the ground prior to planting thecrop. These chemicals are extremely toxic to organisms besides nematodesand many of them may pose serious threats to the environment. With therenewed emphasis on clean water and air by environmental groups andgovernmental agencies, and the detection of many of these activeingredients or the metabolites thereof in ground water and severalnon-target organisms, there has been serious concern as to themanufacture and/or use of these chemicals. One of the most effective,economical, and widely used nematocides, DBCP(1,2-dibromo-3-chloropropane), found in ground water has been judged toinduce male sterility and possible carcinogenesis. Another widely usedchemical, EDB (ethylene dibromide), has also been found in ground water.

[0006] Yet another very common insecticide-nematocide, aldicarb(2-methyl-2-(methylthio)-propionaldehyde-O-(methylcarbamoyl)oxime), hasbeen found to be acutely toxic. Aldicarb has been found in ground waterin several regions of United States. Carbofuran(2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) and 1, 3-D(1,3-dichlorpropane), two very commonly used nematocides, are underspecial review by EPA because of their avian toxicity and possiblecarcinogenic effects. More recently, the decision by EPA to limit andeventually discontinue the use of the soil fumigant, methyl bromide, foragricultural purposes presents a threat to the efficiency and quality ofagricultural production in the United States.

[0007] Natural isolates such as N-acetyl-D-glucosamine, which may bederived from microorganisms which are the waste products of industrialfermentation processes, have been disclosed as nematocidal in U.S. Pat.No. 5,057,141. Biopesticides have been developed as an alternative tochemical pesticides. They are obtained by fermentation and can be usedeither as crude biomass or purified. Typically, fermentations arecarried out at temperatures in the range of 20-40° C. For example,submerged fermentation at 28-30° C. of Paecilomyces fumosoroues fungalisolate ATCC No. 20874 produces fungal biomass for control of nematodeinfestation as disclosed in U.S. Pat. No. 5,360,607; whole fermentationbroth from fermentation at 28° C. of Streptomyces thermoarchaensis NCIB12015 is disclosed as nematocidal in U.S. Pat. No. 5,182,207;fermentation broth obtained from fermentation of Streptomycescyaneogriseus noncyanogenus NRRL 15773 at 28° C. is effective againstnematodes as disclosed in U.S. Pat. No. 5,439,934; and fermentationbroth obtained by fermentation of the fungus Myrothecium verrucaria attemperatures of from 25 to 30° C. is disclosed as nematocidal in U.S.Pat. No. 5,051,255.

[0008] Heating of an infested biomass, as disclosed in U.S. Pat. No.4,229,442, at a temperature of at least 125° C. with water in an amountranging from 1-5 times the dry weight of the biomass, may combatnematodes.

[0009] However, there is still a need for the development of new andeffective nematocides. It is therefore an object of this invention toprovide a method for the production of nematocidal compositions whichare effective and inexpensive.

BRIEF SUMMARY OF THE INVENTION

[0010] The invention is directed to a method of producing a nematocidefrom fermentation broths of microorganisms by heating a fermentationbroth to a temperature of at least about 100° C. after adjusting the pHto about pH 2 or below, or about pH 8 or above. The nematocide thusproduced can be used to prevent plant damage and/or limit the growth ofnematodes.

[0011] The present invention is directed to a method for improvingbiopesticidal activity of materials produced by fermentation comprisingthe steps of:

[0012] fermenting a bacterium or fungus to obtain a fermentation broth;

[0013] adjusting the pH of said fermentation broth to a pH of belowabout 2.5 with a biologically acceptable acid, or to a pH of above about8 with a biologically acceptable alkali or base; and

[0014] heat treating said fermentation broth to a temperature of atleast about 100° C. for at least about 15 minutes, then cooling toambient temperature to obtain a pH-adjusted, heat treated compositionhaving improved biopesticidal activity;

[0015] with the proviso that said fungus is not Myrothecium verrucariawhen the pH of said fermentation broth is adjusted to a pH of belowabout 2.5.

[0016] The invention is also directed to a composition comprising

[0017] a heat-treated, pH adjusted fermentation broth of a fungus orbacterium selected from the group consisting of Gibberella fujikuroi,Streptomyces erythraeus, Bacillus sphaericus, Bacillus thuringiensis andFusarium moniliforme;

[0018] wherein said broth is pH adjusted either to a pH of below about2.5 with a biologically acceptable acid, or to a pH of above about 8with a biologically acceptable alkali or base;

[0019] and wherein said broth is heated to a temperature of at leastabout 100° C. for at least about 15 minutes, then cooled to ambienttemperature.

[0020] The invention is also directed to a method for control ofnematodes on plants which comprises the step of administering to thelocus, soil or seed of plants in need of such treatment, a nematocidallyeffective amount of a composition formed by heating a pH adjustedfermentation broth from a fungus or bacterium;

[0021] wherein said broth is adjusted to a pH of below about 2.5 with abiologically acceptable acid, or to a pH of above about 8 with abiologically acceptable alkali or base;

[0022] and wherein said broth is heated to at least 100° C. for at least15 minutes;

[0023] with the proviso that said fungus is not Myrothecium verrucariawhen the pH of said fermentation broth is adjusted to a pH of belowabout 2.5.

[0024] The composition may be administered at a rate of from about 1 toabout 200 pounds per acre.

[0025] The invention is also directed to a method for producing anematocidal composition comprising the steps of:

[0026] a) fermenting a bacterium or fungus to obtain a fermentationbroth;

[0027] b) suspending said broth in an aqueous solution;

[0028] c) adjusting the pH of said broth in aqueous solution to a pHbelow about pH 2.5 with a biologically acceptable acid;

[0029] d) heating the pH-treated broth of step c) to a temperature of atleast about 100° C. for at least about 15 minutes, then cooling toambient temperature; and,

[0030] e) recovering said composition;

[0031] with the proviso that said fungus is not Myrothecium verrucaria.

[0032] Preferably, the pH is adjusted below about 2; and mostpreferably, the pH is adjusted below about 1.5.

[0033] The pH of the composition formed in step d) may be adjusted to arange of about pH 4 to about pH 8. The fermentation broth of step a) mayhave water-soluble biomass and water-insoluble biomass; and thewater-soluble biomass may be separated from said water-insolublebiomass, and the water-insoluble biomass may be suspended in aqueoussolution in step b).

[0034] The invention is also directed to a method for producing anematocidal composition comprising the steps of:

[0035] a) fermenting a bacterium or fungus to obtain a fermentationbroth;

[0036] b) suspending said broth in an aqueous solution;

[0037] c) adjusting the pH of said broth in aqueous solution to a pHabove about pH 8 with a biologically acceptable alkali or base;

[0038] d) heating the pH-treated broth of step c) to a temperature of atleast about 100° C. for at least about 15 minutes, then cooling toambient temperature;

[0039] e) recovering said composition.

[0040] Preferably, the pH is adjusted above about 9 and most preferablythe pH is adjusted above about 10.

[0041] The pH of the composition formed in step d) may be adjusted to arange of about pH 4 to about pH 8. The fermentation broth of step a) mayhave water-soluble biomass and water-insoluble biomass; and thewater-soluble biomass may be separated from said water-insolublebiomass, and the water-insoluble biomass may be suspended in aqueoussolution in step b).

[0042] For the practice of any aspect of this invention, at least onecompound such as herbicides, antimicrobials, fungicides, insecticides,plant growth regulators or nutrients may be added to the heat-treated,pH adjusted compositions.

DETAILED DESCRIPTION OF THE INVENTION

[0043] To produce a nematocidal preparation, a substantial amount ofbiomass is prepared by submerged fermentation of a bacterium or fungalmicroorganism. Preferred examples of microorganisms include Baccillusspp., Myrothecium spp., Gibberella spp., Streptomyces spp., and Fusariumspp. More preferred are the Myrothecium spp. and the Bacillus spp.

[0044] More specifically, the composition can be obtained fromGibberella fujikuroi (ATCC 12616 or 14164), Streptomyces erythraeus(also known as Saccharopolyspora erythraea, ATCC 11635 or 31772),Bacillus sphaericus (ATCC 4978), Bacillus thuringiensis such as Bacillusthuringiensis israelensis (ATCC 35646), Bacillus thuringiensis kurstaki(ATCC 33679) and Bacillus thuringiensis Berliner (ATCC 19268) andFusarium moniliforme (ATCC 10052). The composition can be obtained fromany bacterium or fungal microorganism, since the method breaks down thecell wall of the microorganism. It is believed that the cell walls ofmicroorganisms (branched polysaccharides) are partially or completelyhydrolyzed under the conditions described herein, resulting in theformation of nematocidal hydrolysis products. Therefore, the method isnot microorganism-specific.

[0045] The material is suspended or dissolved in an aqueous solution,preferably water, and is hydrolyzed by acidification to a pH below aboutpH 2.0 by means of a biologically acceptable acid such as, for example,sulfuric, hydrochloric or phosphoric acid, or organic acids such asacetic or formic acid, and heated at a temperature of at least about100° C. for a period of at least about fifteen minutes. The materialthat is treated in this manner is preferentially the water-insolubleportion of the biomass from the fermentation, and the insoluble materialmay be separated from the water-soluble portion of the fermentationproduct, but separation is not necessary. The entire fermentation brothproduced in the fermentor may be treated by the process of thisinvention. After the mixture has been acidified and heated, it willoften be appropriate to readjust the pH of the mixture to a more neutralpH to enhance safety of handling and to reduce the risk of damage toplants which would be treated with the preparation. Said pH adjustmentmay be carried out by addition of any biologically-acceptable alkali orbase, such as sodium hydroxide, potassium hydroxide, magnesium oxide,magnesium hydroxide, calcium oxide, calcium hydroxide, or ammoniasolution (ammonium hydroxide).

[0046] Alternatively, the treatment can also be achieved by raising thepH to above about pH 8 by means of alkalization withbiologically-acceptable alkali or base such as, for example, sodiumhydroxide, potassium hydroxide, magnesium oxide, magnesium hydroxide,calcium oxide, calcium hydroxide, or ammonia solution (ammoniumhydroxide), then heating the resulting mixture as above. Again, afterheating, it may be appropriate to adjust the pH of the resulting mixtureto a more neutral pH by means of a biologically acceptable acid such as,for example, sulfuric, hydrochloric or phosphoric acid, or organic acidssuch as acetic or formic acid.

[0047] The heating step may be performed at a pressure above atmosphericpressure, if necessary. For example, elevated pressure may be achievedby heating within an autoclave.

[0048] The product thus produced is used to protect plants or controlthe growth of nematodes by applying it in solid form or as a suspensionin aqueous solution, preferably water, directly to the surface or theroot zone of the soil in which the plants are grown.

[0049] An advantage of the method which we have discovered is that thenematocidal composition is inexpensive and safe. The materials employedin the process include the fermentation broth, comprising thewater-insoluble solids contained in such broth, which may be wastesolids from any industrial fermentation process, such as a fermentationcarried out to prepare pharmaceutical or agricultural products, or foodsor beverages, and ordinary acids and bases. Previously employedindustrial processes for production of nematocidal preparations arechemical syntheses which use dangerous and toxic starting materials andresult in waste streams of high toxicity which must be disposed of.

[0050] The method involves a post-treatment of a fermentation material,which can be industrial fermentation waste, to produce a nematocidalcomposition.

[0051] As used herein the terms “nematocide” or “nematocidal”, and thephrases “prevent plant damage” and “control of growth”, with respect tonematodes, include not only the rapid, direct killing of nematodes, butalso the concept of repelling nematodes, the prevention or effectivecontrol of their multiplication or reproduction, the prevention ofnematode egg hatching, and confusing or immobilizing the nematodes sothat they are prevented from finding a mate or a plant to parasitize.

[0052] The methods of using the compositions of this invention fornematode control are by application to any field, fruit, vegetable,floral or ornamental crop or nursery crop that is sensitive to attack byplant parasitic nematodes, particularly the Meloidogyne species. Methodsof application are well-known in the art and include direct applicationto the soil, either as a liquid or a dried solid, controlled release ofthe bioactive components from solid formulations into the surroundingsoil, application to the plant roots directly before planting in thesoil, foliar application and the like.

[0053] The term “soil”, used herein is intended to include all mediacapable of supporting the growth of plants and may include humus, sand,silt, loam, manure, compost and commercial potting mixtures amongothers.

[0054] The term “fermentor”, as used herein refers to apparatus used forvarious types of fermentation methods including, but not limited to,shaken culture, solid-state, continuous and batch fed methods that arecontemplated for production of the fermentation broths of this inventionin both laboratory and large scale fermentation processes.

[0055] The term “biologically acceptable acid”, as used herein refers toacids such as sulfuric acid, phosphoric acid, hydrochloric acid, aceticacid or formic acid.

[0056] The term “biologically acceptable alkali or base”, as used hereinrefers to bases such as sodium hydroxide, potassium hydroxide, magnesiumoxide, magnesium hydroxide, calcium oxide, calcium hydroxide or ammoniumhydroxide.

[0057] The process of this invention may utilize various media for theinitial culture growth and can consist of potato-dextrose agar, hayinfusion agar, corn meal agar, leaf litter agar, PCNB agar, soil infuson (modified), or Yeast Malt Agar as are defined in the Manual ofIndustrial Microbiology and Biotechnology, Demain and Solomon, AmericanSociety of Microbiology, Washington, D.C., 1986.

[0058] According to one embodiment of this invention, fermentation iscarried out in shake-flasks or in stationary-vat fermentors. Inshake-flasks, aeration is provided by agitation of the flask whichcauses mixing of the medium with air. In the stationary fermentors,agitation is provided by impeller means such as a disc turbine, vaneddisc, open turbine, or marine propeller; and aeration is accomplished byinjecting air or oxygen into the fermentation mixture.

[0059] The fermentation medium consists of suitable sources of carbon,nitrogen, inorganic salts, and growth factors assimilable by themicroorganism. Suitable examples of carbon sources are various sugarssuch as dextrose, glucose, lactose, and maltose, starch, dextrin, cornmeal and glycerol.

[0060] The sources of nitrogen can be of organic, inorganic or mixedorganic/inorganic origin. Examples of nitrogen sources that can be usedin the culture medium are soybean meal, corn steep liquor, peanut meal,cottonseed meal, corn germ meal, fish meal, lard water, and variousammonium salts.

[0061] The inclusion of certain amounts of minerals and growth factorsin the fermentation medium is also helpful. Crude medium ingredientssuch as distillers' solubles, corn steep liquor, fish meal, yeastproducts, peptonized milk and whey contain not only minerals but growthfactors. However, inorganic salts such as potassium phosphate, sodiumchloride, ferric sulfate, calcium carbonate, cobalt chloride, magnesiumsulfate, and zinc sulfate can be added to the fermentation medium.

[0062] Solid materials, such as calcium carbonate may be added in thisprocess, to help with pH control, which sometimes favors particulartypes of pellet formation for best results.

[0063] The process of producing the fermentation materials for use inthis invention, while utilizing a shaken culture fermentation techniquemay also use such a technique for the initial stages or inoculumproduction as well. Production cultures are started from specially growninocula. Growth is generally rapid at first. It then slows down and astationary phase is usually reached. The production yield depends on thequantity of cells present, their specific activity, and the span oftheir product-forming capacity.

[0064] The inoculum is placed in a liquid medium which is selectedempirically for its ability to allow the recovery of the majority of thecells in the population. The spores produced on an initial growthmedium, such as potato-dextrose agar, are transferred into growth mediumcontained in a flask (termed seed flask), that would allow for thegermination and initial growth of culture. The germinated spores in anactive growth state are then transferred to Erlenmeyer flasks(shake-flasks) or stationary-vat fermentors containing the specificfermentation medium. A 1-2% inoculum is typically produced for thefermentation stage of development.

[0065] The inoculum medium is within the purview of those skilled in theart, and additional information may be found in the Manual of IndustrialMicrobiology and Biotechnology, pages 31-40, supra.

[0066] A wide range of shaker-culture apparatus may be used in thepractice of this invention. The main types of apparatus are based oneither rotary or reciprocating shaking machines. The process hereinpreferably uses rotary shakers in which the flasks move in orbits ofabout 50 mm at about 200 to about 250 rpm, (but may vary between 100 and500 rpm). The culture moves smoothly around the inside of the flask(which is usually an Erlenmeyer flask). The scale-up of the fermentationprocess is well known to those skilled in the art.

[0067] The purpose of shaking in submerged culture is to supply oxygenand nutrients to the growing cells. In shaken cultures, the medium inthe fermentation flasks is inoculated with cells or spores, as is thecase herein. The strain used as an inoculum is held as a master culture,in the freeze-dried state or at reduced temperatures, such as −70° C.The optimal spore concentration to be used for the inoculum is easilydetermined by those skilled in the art by routine experimentation.

[0068] The biopesticidal compositions of the present invention can beused against plant parasitic nematodes, including, for example,Meloidogyne spp., Pratylenchus spp.,

[0069] Radopholus similis, Ditylenchus dipsaci, Heterodera spp.,Xiphinema spp., Globodera spp. and Hoplolaemus spp.

[0070] The processed fermentation materials prepared according to theprocess of this invention can be used to control nematodes for a varietyof agricultural applications on many different plants and fruitsincluding, but not limited to, artichokes, aubergines, banana, barley,beet roots, cacao, carrots, cassava, celery, chickpea, citrus, coconut,coffee, cole crops, corn, cotton, cowpea, eggplant, field bean, forages,ginseng, grape, guava, various lettuces, melons, millet, oat, okra,ornamentals, papaya, peanut, pepper, pigeon pea, pineapple, potatoes,rice, rye, sorghum, soybean, sugar beet, sugar cane, sweet peppers,sweet potato, tea, tobacco, tomatoes, turf, wheat and yam. Cultivatedflowers can be protected according to the present invention, such ascarnations, rose bushes, gerberas, chrysanthemums, pot plants,philodendrons, ferns, figs, pothos, sanseverias, and cacti; examples ofnursery plants would include all the ornamental and flowering shrubs.

[0071] The bioactive materials can be incorporated into the soil offlower pots or containers, by direct application to the area to betreated at the time of planting, or up to several days earlier, or byapplication in a controlled release form. Application to field ororchard crops can be by granule dispersement on the surface withturnover of the soil by a claw cultivar or a light plow, generally toabout 10 cm up to about 20 cm depth of soil. As the nematocide is watersoluble, a drip irrigation method for application is also possible.

[0072] The compositions of the present invention can be in a suitableform for direct application or as a concentrate or primary compositionwhich requires dilution with a suitable quantity of water or otherdiluent before application. The pesticidal concentration will varydepending upon the nature of the particular formation, specificallywhether it is a concentrate or to be used directly.

[0073] The nematocidally effective amount of the active materials willdepend upon the population of the nematode expected to be encountered,the nematode type, soil, crop, and moisture. In general, the compositionmay be applied at a field rate of from about 1 to about 200/lbs peracre; preferably at a rate of from about 5 to about 100/lbs per acre andmost preferably at a rate of from about 10 to about 60 lbs/acre. Thenematocidal compositions may be in the form of a suspension, a solution,an emulsion, a dusting powder, a dispersible granule, a wettable powder,an emulsifiable concentrate, an aerosol or impregnated granule,formulated by techniques well known to those skilled in the art.

[0074] Additives to these compositions may include surface activeagents, inert carriers, preservatives, humectants, feeding stimulants,attractants, encapsulating agents, binders, emulsifiers, dyes, U.V.protectants, buffers, flow agents, or other components which facilitateproduct handling and application for protection against nematodes.

[0075] Examples of inert carriers include inorganic minerals such askaolin, mica, gypsum, fertilizer, phyllosilicates, carbonates, sulfates,or phosphates; organic materials such as sugar, starches orcyclodextrins; or botanical materials such as wood products, cork,powdered corn cobs, rice hulls, peanut hulls and walnut shells.

[0076] Suitable surface active agents include anionic compounds such ascarboxylates, for example an alkali metal carboxylate of a long chainfatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acidwith fatty alcohol ethoxylates or salts of such esters; fatty alcoholsulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate orsodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylatedalkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkylaryl sulfonates such as alkyl benzene sulfonates or lower alkylnaphthalene sulfonates such as butyl naphthalene sulfonate; salts orsulfonated naphthalene-formaldehyde condensates; salts of sulfonatedphenol-formaldehyde condensates or more complex sulfonates such as theamide sulfonates. Non-ionic agents include condensation products offatty acid esters, fatty alcohols, fatty acid amides or fatty alkyl- oralkenyl-substituted phenols with ethylene oxide, fatty esters ofpolyhydric alcohol ethers, such as sorbitan fatty acid esters,condensation products of such esters with ethylene oxide, such aspolyoxyethylene sorbitan fatty acid esters, block copolymers of ethyleneoxide and propylene oxide, and acetylenic glycols. Examples of cationicsurface active agents include an aliphatic mono-, di-, or polyamine asan acetate, naphthenate or oleate; an oxygen containing amine such as anamine oxide of polyoxyethylene alkylamine; an amide-linked amineprepared by the condensation of a carboxylic acid with a di- orpolyamine; or a quaternary ammonium salt.

[0077] It is also contemplated that the materials of this invention maybe used in combination with other essential biologicals or beneficialmicroorganisms or active ingredients, such as herbicides,anti-microbials, fungicides, insecticides, plant growth regulators ornutrients.

[0078] The compositions of this invention may also be formulated asactive mixtures which may include finely divided dry or liquid diluents,extenders, fillers, conditioners, and excipients, including variousclays, diatomaceous earth, talc and the like, or water and variousorganic liquids and mixtures thereof.

[0079] Of course, the present invention is not limited to the particularembodiments and modes of operation described herein and it is possibleto imagine a number of variations in the details without departing fromthe scope of this invention.

[0080] The examples below are presented to describe preferredembodiments and utilities of the invention and are not meant to limitthe invention unless otherwise stated in the claims appended hereto.

EXAMPLE 1

[0081] Water-insoluble materials were isolated from a Myrotheciumverrucaria fermentation broth by centrifuging. These solids were washedby re-suspending in distilled water and centrifuged again. The washingprocess was repeated twice more, and the solids were recovered in powderform by freeze-drying. This powdered material was subsequently treatedby suspending a weighed amount of it in water and heating only, orheating within several acidic environments (as detailed in Table 1below). The materials resulting from these conditions were readjusted topH 4, freeze dried into a powder, and tested in a contact assay withRKN. All heat treatments were carned out at 121° C. for four hours in anautoclave. Groups of approximately 50 nematodes for each replicate wereincubated in 12½% aqueous suspensions of each of the reconstitutedmaterials for a period of 24 hours, then counted to determine thepercent mortality of RKN. The materials resulting from heating underacid conditions of pH 2 or lower showed significant increases innematocidal activity. TABLE 1 Contact Nematocidal Assay for FermentationSamples (Insoluble Fermentation Fraction) Sample Treatment Average %Mortality of RKN¹ A Heated 5 B pH 3 + Heated 7 C pH 2 + Heated 88 D pH1.5 + Heated 100 E pH 1.25 + Heated 100 F Untreated 5

EXAMPLE 2

[0082] Filter cakes (washed, damp fermentation solids) from two fungalfennentations were re-suspended in three times their weight of water,adjusted to pH 2.0 with sulfuric acid and heated in an autoclave at 121°C. for three hours. The samples were cooled, re-adjusted to pH 4.05-4.10with sodium hydroxide solution and made up with distilled water to afinal weight eight times that of the cake used (12.5% concentration).Groups of approximately 50 root-knot nematodes for each replicate wereincubated for 24 hours in these suspensions as well as a distilled watercontrol and counted to determine mortality. The heat-treated fungalfermentation solids showed strong nematocidal activity, as indicated inTable 2. TABLE 2 Nematodes Exposed to Average Percent Mortality sterilewater 0 heated Streptomyces erythraeus solids 97.2 heated Gibberellafujikuroi solids 98.5

EXAMPLE 3

[0083] Dry material was recovered by lyophilization from a suspension offermentation solids from an unclassified Streptomyces species. The solidmaterial (4.56 g) was re-suspended in 40 mL of water, adjusted to pH 1.9with sulfuric acid and heated at 121-122° C. for three hours. Aftercooling, the pH was re-adjusted to 4.0 with sodium hydroxide solution,giving a final suspension containing 9% by weight of the fermentationsolids. Groups of approximately 50 root-knot nematodes for eachreplicate were incubated for 24 hours with this suspension or with anantibiotic control solution (100 units of penicillin plus 0.1 mg ofstreptomycin per mL) and then counted to determine mortality. Theheat-treated fermentation solid suspension was effective in killing theplant-parasitic nematodes, as shown in Table 3. TABLE 3 Average PercentNematodes Exposed to Mortality Antibiotic control 0.4 Heated suspensionof Streptomyces solids 100

EXAMPLE 4

[0084] Water-insoluble materials were isolated from a Myrotheciumfermentation broth by centrifuging as described in Example 1. Thisinsoluble fraction was acid treated by suspending a weighed amount inwater and adjusting to pH 1.72. The sample was divided and one portionwas heated at 120° C. for three hours while the other portion was keptat room temperature. The materials were both readjusted to pH 4 andlyophilized into a powder. The powders were reconstituted and used in atest measuring percent mortality of root knot nematodes. Nematodes wereincubated in solution for 24 hours, then counted to determine living anddead nematodes, as shown in Table 4. Reducing the pH to 1.72 and heatingincreased the percent mortality of root-knot nematodes. TABLE 4 Effectsof pH changes on contact nematocidal activity Avg. % Sample TreatmentMortality² Insoluble fermentation Heated at pH 1.72 96.3 fractionInsoluble fermentation Unheated at pH 1.72 <1% fraction - Control

EXAMPLE 5 Effects of Acid Digestion on Nematocidal Activity ofFermentation Broths (Greenhouse Evaluation)

[0085] Whole myrothecium fermentation culture broth was divided into twoportions. One portion was treated by reducing the pH to 1.5 and heatingat 121° C. for two hours, then readjusting the pH to 4. The otherportion was an untreated control. The resulting materials werefreeze-dried into a powder. The powders were reconstituted in water toequal concentrations by weight and dosed at a rate of 1.0 gram/pot ontosmall soil pots containing cucumber seedlings and approximately 800root-knot nematodes per pot. The plants were maintained with light andwater for one week, then removed from the soil. The galls (rootswellings resulting from infection by root-knot nematode, Meloidogyneincognita) on each root were counted to determine the rate of nematodeinfection. Efficacy is measured by the percent gall reduction from theuntreated, nematode infected control plants. As shown in Table 5, acidtreated and heated fermentation broth was effective at reducing thenematode infection by about 80%, whereas fermentation broth that was notacid treated and heated controlled about 30% of the galling. TABLE 5Sample % Gall Control Fermentation Broth (FB) 29.46 Acid Treated FB80.62

[0086] The above materials were also tested in a contact assay whereroot-knot nematodes were incubated for 24 hours in reconstitutedsolutions made from the lyophilized powders. Material that was acidtreated and heated increased the percent mortality of the nematodes,whereas the untreated broth and broth that was heated without the acidtreatment showed low mortality of the nematodes, as shown in Table 6.TABLE 6 Average % Treatment of RKN Mortality Fermentation Broth -unmodified 3.5 Fermentation Broth - heated 3.4 Fermentation Broth -heated under 100 acidic conditions

Example 6 Nematocidal Activity of Acid-or Alkali Digested FermentationSamples (Contact Assay)

[0087] Myrothecium fermentation broth was heated in acid and basic(alkaline) conditions, and the efficacy of the resulting materials wascompared in a nematocidal assay (% mortality of root knot nematodes).The control broth sample was adjusted to neutral pH (pH 7), and othersamples were adjusted to acidic (pH 2) and basic (pH 12) conditions.Half of each sample was heated at 121° C. for two hours. Once cooled,all the samples were readjusted to neutral pH and lyophilized (dried toa powder). The powders were reconstituted in water to equalconcentrations by weight and tested in a contact assay. The contactassay consisted of RKN incubated for 24 hours in five concentrations ofeach material, with multiple replications. The nematodes were counted,and the number of dead nematodes determined for each concentration forevery sample. Data from this test was analyzed by PROBIT analysis todetermine the LC₅₀ (lethal concentration or dose to obtain an estimated50% mortality of the nematodes within 95% confidence range).Fermentation broth when heated in either acid or basic conditions showedincreased toxicity to nematodes, indicated by a lower LC₅₀ value forthese materials, as shown in Table 7. Materials that were pH adjustedbut not heated did not show a significant increase of toxicity tonematodes. TABLE 7 LC₅₀ against M. incognita Sample Unheated HeatedFermentation Broth (FB) 3.59 5.54 pH 7 FB acid condition (pH 2) 3.581.01 FB alkaline condition 2.52 0.88 (pH 12)

[0088] When plant parasitic nematodes are placed in direct contact withthe compositions so produced, the nematodes are paralyzed or killed,depending on the concentration of the composition applied. When the soilin which potted plants are growing is treated with a suspension of theproduct produced according to the method of this invention, and theninoculated with plant parasitic nematodes, after a growing period ofseveral days, the plants are healthier, with larger and heavier aerialparts than untreated plants similarly inoculated with nematodes. Theroots of the treated plants also exhibited fewer root galls (symptoms ofnematode attack and invasion) than the untreated plants. In the field,the composition produced may be applied as a suspension in water or as adry granular material to the plants to be protected from parasiticnematodes.

Example 7

[0089] Portions of whole fermentation beer from the submergedfermentation of Myrothecium sp. were adjusted to pH's of 4, 8, 9, 10 or1 (+0.1) with sulfuric acid or sodium hydroxide and heated in anautoclave for two hours at 121° C. Control samples were pH adjusted butnot heated. After heating, the samples were cooled to room temperature,and all samples were adjusted to pH 4.1 (±0.1) using sulfuric acid. Thensamples were diluted to a final concentration of 2% solids. Groups ofMeloidogyne incognita (root-know nematodes, RKN) were incubated in thesuspensions as well as a dilute antibiotic control solution (1 unit/mLof penicillin and 0.1 mg/mL of streptomycin), and after 24 hours liveand dead individuals were counted. Beer samples heated at elevated pHdemonstrated increased nematocidal activity over non-heated samples, asshown in Table 8. TABLE 8 Whole-Beer Alkaline Heat Treatment PercentMortality of RKN at 2% Concentration Heated Non-heated pH 4 8.3 5.7 pH 86.4 0.0 pH 9 18.5 1.8 pH 10 33.3 0.0 pH 11 32.3 2.1 antibiotic controlNA 0.0

All References Cited are Hereby Incorporated by Reference.

[0090] The present invention is illustrated by way of the foregoingdescription and examples. The foregoing description is intended as anon-limiting illustration, since many variations will become apparent tothose skilled in the art in view thereof. It is intended that all suchvariations within the scope and spirit of the appended claims beembraced thereby.

[0091] Changes can be made in the composition, operation and arrangementof the method of the present invention described herein withoutdeparting from the concept and scope of the invention as defined in thefollowing claims:

We claim:
 1. A method for improving biopesticidal activity of materialsproduced by fermentation comprising the steps of: fermenting a bacteriumor fungus to obtain a fermentation broth; adjusting the pH of saidfermentation broth to a pH of below about 2.5 with a biologicallyacceptable acid, or to a pH of above about 8 with a biologicallyacceptable alkali or base; and heat treating said fermentation broth toa temperature of at least about 100° C. for at least about 15 minutes,then cooling to ambient temperature to obtain a pH-adjusted, heattreated composition having improved biopesticidal activity; with theproviso that said fungus is not Myrothecium verrucaria when the pH ofsaid fermentation broth is adjusted to a pH of below about 2.5.
 2. Themethod of claim 1 wherein said fungus or bacterium is selected from thegroup consisting of Gibberella fujikuroi, Streptomyces erythraeus,Bacillus sphaericus, Bacillus thuringiensis and Fusarium moniliforme. 3.A composition comprising a heat-treated, pH adjusted fermentation brothof a fungus or bacterium selected from the group consisting ofGibberella fujikuroi, Streptomyces erythraeus, Bacillus sphaericus,Bacillus thuringiensis and Fusarium moniliforme; wherein said broth ispH adjusted either to a pH of below about 2.5 with a biologicallyacceptable acid, or to a pH of above about 8 with a biologicallyacceptable alkali or base; and wherein said broth is heated to atemperature of at least about 100° C. for at least about 15 minutes,then cooled to ambient temperature.
 4. A method for control of nematodeson plants which comprises the step of administering to the locus, soilor seed of plants in need of such treatment, a nematocidally effectiveamount of a composition formed by heating a pH adjusted fermentationbroth from a fungus or bacterium; wherein said broth is adjusted to a pHof below about 2.5 with a biologically acceptable acid, or to a pH ofabove about 8 with a biologically acceptable alkali or base; and whereinsaid broth is heated to at least 100° C. for at least 15 minutes; withthe proviso that said fungus is not Myrothecium verrucaria when the pHof said fermentation broth is adjusted to a pH of below about 2.5. 5.The method of claim 4 wherein said composition is administered at a rateof from about 1 to about 200 pounds per acre.
 6. The method of claim 4wherein said fungus or bacterium is selected from the group consistingof Gibberella fujikuroi, Streptomyces erythraeus, Bacillus sphaericus,Bacillus thuringiensis and Fusarium moniliforme.
 7. The method of claim4 wherein said composition further comprises at least one compoundselected from the group consisting of herbicides, antimicrobials,fungicides, insecticides, plant growth regulators and nutrients.
 8. Amethod for producing a nematocidal composition comprising the steps of:a) fermenting a bacterium or fungus to obtain a fermentation broth; b)suspending said broth in an aqueous solution; c) adjusting the pH ofsaid broth in aqueous solution to a pH below about pH 2.5 with abiologically acceptable acid; d) heating the pH-treated broth of step c)to a temperature of at least about 100° C. for at least about 15minutes, then cooling to ambient temperature; and, e) recovering saidcomposition; with the proviso that said fungus is not Myrotheciumverrucaria.
 9. The method of claim 8 wherein said pH is adjusted belowabout
 2. 10. The method of claim 8 wherein said pH is adjusted belowabout 1.5.
 11. The method of claim 8 further comprising adjusting pH ofsaid composition formed in step d) to a range of about pH 4 to about pH8.
 12. The method of claim 8 further comprising adding at least onecompound to said composition of step e), said compound selected from thegroup consisting of herbicides, antimicrobials, fungicides,insecticides, plant growth regulators and nutrients.
 13. The method ofclaim 8 wherein said fermentation broth of step a) has water-solublebiomass and water-insoluble biomass.
 14. The method of claim 13 whereinsaid water-soluble biomass is separated from said water-insolublebiomass, and said water-insoluble biomass is suspended in aqueoussolution in step b).
 15. A method for producing a nematocidalcomposition comprising the steps of: a) fermenting a bacterium or fungusto obtain a fermentation broth; b) suspending said broth in an aqueoussolution; c) adjusting the pH of said broth in aqueous solution to a pHabove about pH 8 with a biologically acceptable alkali or base; d)heating the pH-treated broth of step c) to a temperature of at leastabout 100° C. for at least about 15 minutes, then cooling to ambienttemperature; e) recovering said composition.
 16. The method of claim 15wherein said pH is adjusted above about
 9. 17. The method of claim 15wherein said pH is adjusted above about
 10. 18. The method of claim 15further comprising adjusting pH of said composition formed in step d) toa range of about pH 4 to about pH
 8. 19. The method of claim 15 furthercomprising adding at least one compound to said composition of step e),said compound selected from the group consisting of herbicides,antimicrobials, fungicides, insecticides, plant growth regulators andnutrients.
 20. The method of claim 15 wherein said fermentation broth ofstep a) has water-soluble biomass and water-insoluble biomass.
 21. Themethod of claim 20 wherein said water-soluble biomass is separated fromsaid water-insoluble biomass, and said water-insoluble biomass issuspended in aqueous solution in step b).